Insects and other pests cost farmers billions of dollars annually in crop losses and expense to keep these pests under control. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and to home gardeners. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Insect pests are mainly controlled by intensive applications of chemical pesticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Good insect control can thus be reached, but these chemicals can sometimes also affect other beneficial insects. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect populations. This has been partially alleviated by various resistance management practices, but there is an increasing need for alternative pest control agents. Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing pesticidal toxins like delta-endotoxins, have also been applied to crop plants with satisfactory results, offering an alternative or compliment to chemical pesticides. The genes coding for some of these delta-endotoxins have been isolated and their expression in heterologous hosts have been shown to provide another tool for the control of economically important insect pests. In particular, the expression of insecticidal toxins, such as Bacillus thuringiensis delta-endotoxins, in transgenic plants have provided efficient protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce applications of chemical insect control agents.
The soil microbe Bacillus thuringiensis is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. Bacillus thuringiensis continues to be the leading source of novel insecticidal proteins for development of plant incorporated pesticides. Using various strains of bacterial isolates, we have invented new Bt toxins that are active against commercially important insect pests. In the North American maize insect resistance market, Spodoptera frugiperda (fall armyworm “FAW”), Ostrinia nubialis Hübner (European corn borer “ECB”), and Helicoverpa zea Boddie (corn earworm “CEW”) are the key driver pests, although there are other key insect pests in other geographies (e.g. Helicoverpa armigera (cotton bollworm “CBW” or corn earworm “CEW”)) and additional secondary, but important insect pest species. Bt toxins represent over 90% of the bioinsecticide market and essentially the entire source of genes for transgenic crops that have been developed to provide resistance to insect feeding. Bt bacteria produce insecticidal delta-endotoxins including Crystal (Cry), Cytotoxin (Cyt), and Vegetative Insecticidal Protein (VIP) toxins, depending on their gene and protein structure. Cry toxins are produced during spore formation as insoluble crystal proteins. VIP toxins, on the other hand, are produced as soluble proteins during the vegetative stage of Bt bacterial growth. VIP proteins are distinct from Cry proteins in their structure, but share the property with Cry toxins of being pore formers acting on cells located in the insect midgut (Yu, C.-G., et al., 1997 Appl. Environ. Microbiol. 63:532-536, Lee, M. K., et al., 2003, Appl. Environ. Microbiol. 69: 4648-4657, Shotkoski, F., et al., 2003, Proc. Beltwide Cotton Conf, 89-93). We describe here the invention of new VIP toxins that have broad spectrum insecticidal activity, including insecticidal activity against ECB, which is unique compared to VIP proteins currently known (Yu, C.-G., et al., 1997 Appl. Environ. Microbiol. 63:532-536).
Patent documents WO2013/134523, WO 94/21795, WO 96/10083, U.S. Pat. Nos. 5,877,012, 6,107,279, 6,137,033, and 6,291,156, as well as Estruch et al. (1996, Proc. Natl. Acad. Sci. 93:5389-5394) and Yu et al. (1997, Appl. Environ. Microbiol. 63:532-536), describe a class of insecticidal proteins called VIP3. VIP3 genes encode approximately 88 kDa proteins that are produced and secreted by Bacillus during its vegetative stages of growth. These toxins were reported to be distinct from crystal-forming delta-endotoxins. These documents make specific reference to toxins designated VIP1A(a), VIP1A(b), VIP2A(a), VIP2A(b), VIP3A(a), and VIP3A(b). See also Lee et al., AEM vol. 69, no. 8 (August 2003), pages 4648-4657, for a discussion of the mechanism of action and truncation of VIP proteins.
The VIP3A protein possesses insecticidal activity against a wide spectrum of lepidopteran pests, including FAW, CEW, Agrotis ipsilon Hufnagel (black cutworm “BCW”), and Heliothis virescens Fabricius (tobacco budworm “TBW”). More recently, VIP proteins have been found to be toxic to certain species of hemipteran insect pests (Nanasaheb, P. et al, Toxins (Basel) vol. 4, no. 6 (June 2012), pages 405-429, Sattar S. and Maiti M. K., J. Microbiol. Biotechnol. 2011, 21:937-946). Thus, the VIP class of proteins display a unique spectrum of insecticidal activities. Other disclosures, WO 98/18932, WO 98/33991, WO 98/00546, and WO 99/57282, have also now identified homologues of the VIP3 class of proteins.
The continued use of chemical and biological agents to control insect pests heightens the chance for insects to develop resistance to such control measures. Also, the high selectivity of biological control agents often results in only a few specific insect pests being controlled by each agent. Despite the success of ECB-resistant transgenic corn, the possibility of the development of resistant insect populations threatens the long-term durability of Cry proteins in ECB control and creates the need to discover and develop new Cry or other types of biological control agents to control ECB and other pests. Insect resistance to Bt Cry proteins can develop through several mechanisms (Heckel et al., 2007, Pigott and Ellar, 2007). Multiple receptor protein classes for Cry proteins have been identified within insects, and multiple examples exist within each receptor class. Resistance to a particular Cry protein may develop, for example, by means of a mutation within the toxin-binding portion of a cadherin domain of a receptor protein. A further means of resistance may be mediated through a protoxin-processing protease. Thus, resistance to Cry toxins in species of Lepidoptera has a complex genetic basis, with at least four distinct, major resistance genes. Lepidopteran insects resistant to Cry proteins have developed in the field within the species DBM (diamondback moth) (Tabashnik, 1994), Trichoplusia ni Hübner (cabbage looper “CL”; Janmaat and Myers 2003, 2005), and CEW (Tabashnik et al., 2008). Therefore development and deployment of new high potency plant incorporated pesticidal proteins such as those disclosed herein are both useful and needed.
Therefore, there remains a need to discover new and effective pest control agents that provide an economic benefit to farmers and that are environmentally acceptable. Particularly needed are control agents targeted to a wide spectrum of economically important insect pests that efficiently control insect populations that are, or could become, resistant to existing insect control agents and those with equal to or increased potency compared to current control agents.